Citrate and low-dose heparin combined anticoagulation in pediatric continuous renal replacement therapy

There remains no optimal anticoagulation protocol for continuous renal replacement therapy (CRRT) with regional citrate anticoagulation (RCA) in pediatric patients with elevated D-dimer levels. We aimed to assess the effects of different anticoagulation strategies on the risk of CRRT filter clotting in these patients. Pediatric patients undergoing CRRT were retrospectively grouped based on pre-CRRT D-dimer levels and anticoagulant: D-RCA group (normal D-dimer, RCA only, n = 22), D+ RCA group (elevated D-dimer, RCA only, n = 50), and D+ RCA+ systemic heparin anticoagulation (SHA) group (elevated D-dimer, RCA combined with SHA, n = 55). The risk of filter clotting and incidence of bleeding were compared among the groups. Among the groups, the D+ RCA+ SHA group had the longest filter lifespan; further, the incidence of bleeding was not increased by concurrent use of low-dose heparin for anticoagulation. Moreover, concurrent heparin anticoagulation was associated with a decreased risk of filter clotting. Contrastingly, high pre-CRRT hemoglobin and D-dimer levels and post-filter ionized calcium level > 0.4 mmol/L were associated with an increased risk of filter clotting. RCA combined with low-dose heparin anticoagulation could reduce the risk of filter clotting and prolong filter lifespan without increasing the risk of bleeding in patients with elevated D-dimer levels undergoing CRRT.


CRRT anticoagulation strategies
A 4% anticoagulant citrate solution (Chengdu Qingshan Likang Pharmaceutical Co., Ltd., Sichuan, China) was used as the anticoagulant in all included patients.The initial blood pump flow rate was 3 mL/kg/min, with the initial citrate flow rate (mL/h) being 1.5× blood pump flow rate (mL/min).The target intracorporeal and postfilter calcium levels were 1-1.2 and 0.2-0.4mmol/L, respectively.From March 2021 onwards, attempts were made to reduce the risk of clotting in the extracorporeal circuit during CRRT in patients with a high clotting risk due to elevated D-dimer levels.After excluding patients with contraindications to heparin and those receiving systemic anticoagulants (including heparin and low-dose heparin) due to other reasons, continuous intravenous infusion of 5 U/kg/h heparin with RCA was administered in patients with D-dimer levels > 0.5 μg/mL.The heparin dose was lowered if the activated partial thromboplastin time (aPTT) exceeded twice the normal aPTT values or the activated clotting time exceeded 180 s.Moreover, heparin was discontinued if the patient experienced a clinical bleeding episode during combination anticoagulation therapy.

CRRT prescription
Indications for CRRT commencement were determined at the discretion of the attending physician at the PICU.CRRT was performed using the Prismaflex device (Gambro, Lund, Sweden), the multiFiltrate multifunctional blood purification system (Fresenius Medical Care, Bad Homburg, Germany), and the corresponding catheter and filter sets of the CRRT equipment.Vascular access was gained by placing a single dual-lumen catheter in the right internal jugular or femoral veins under ultrasound guidance.The initial dialysate and replacement fluid flow rates during CVVHDF were set at approximately 35 mL/kg/h, adopted post-dilution during the RCA-CVVHDF.Calcium-containing, bicarbonate-free hemofiltration and replacement fluid (4 L/bag; Chengdu Qingshan Likang Pharmaceutical Co., Ltd., Sichuan, China) was used as the replacement fluid.Bicarbonate supplementation was not performed during CRRT initiation.Buffer bases were generated after sodium citrate metabolism and adjusted based on the acid-base status of the patient.Calcium supplementation was performed based on intracorporeal ionized calcium; blood calcium levels were monitored during CRRT.Blood gas analysis, including measurement of ionized calcium and post-filter ionized calcium levels, was conducted once every 4-6 h.Routine blood tests, liver and kidney function tests, blood electrolyte level measurement, and comprehensive coagulation profiling were performed once every 6-8 h.Treatment parameters during CRRT were adjusted by titration based on the patient's condition and monitored indicators.

Data collection
Demographic characteristics, clinical data, CRRT parameters, and patient outcomes were recorded.Filter lifespan and the incidence of bleeding episodes during CRRT were set as the primary and secondary outcomes, respectively.A bleeding episode was defined as any clinically visible bleeding or a > 20 g/L decrease in hemoglobin levels during CRRT.

Statistical methods
All statistical analyses were performed using SPSS 25.0 (IBM Corp., Armonk, NY).Normally distributed continuous variables are expressed as mean ± standard deviation (x ± s) and were compared using analysis of variance.Categorical data are expressed as counts (percentages) and were compared using the Chi-squared test.Nonnormally distributed continuous data are expressed as median (quartiles) [M (P25, P75)] and were compared using the Kruskal-Wallis H test. Filter lifespan was analyzed using Kaplan-Meier estimates.Factors influencing

Ethics approval and consent to participate
This study was conducted with approval from the Ethics Committee of Hunan Children's Hospital, approval number HCHLL-2023-192.The requirement for informed consent was waived.

General status of the participants
A total of 127 pediatric patients were included in this study.Figure 1 shows the flowchart of the study population selection process.Among the patients, 22, 50, and 55 patients belonged to the D− RCA only group, D+ RCA only group, and D+ RCA+ SHA group, respectively.In total, 52% of the patients received mechanical ventilation

Filter lifespan and bleeding episodes
The  2. In total, 43% (55/127) of the patients underwent SHA with RCA, and the incidence of bleeding episodes was not increased in these patients.There were no significant among-group differences in the incidence of bleeding episodes (D− RCA only: 18.2% vs. D+ RCA only: 18% vs. D+ RCA+ SHA: 14.5%, P = 0.869).Table 2 summarizes the filter characteristics and patient outcomes.

Discussion
In this retrospective single-center cohort study, we compared the risk of filter clotting and incidence of bleeding episodes among the patients who underwent different anticoagulation therapies during CRRT.Our main finding was that the adoption of the combination of RCA and low-dose heparin anticoagulation strategy during CRRT may prolong the filter lifespan without increasing the incidence of bleeding episodes in patients with elevated D-dimer levels.Therefore, combination anticoagulation therapy may be superior to RCA alone.Anticoagulation therapy is of utmost importance for preventing clotting in the extracorporeal circuit during CRRT.Various anticoagulation strategies, including SHA, RCA, nafamostat mesylate, low-molecular-weight heparin, argatroban, prostaglandin I2, and regional unfractionated heparin, are available for CRRT and have been extensively studied [12][13][14][15][16][17][18] .A meta-analysis that included 37 randomized controlled studies and 2648 participants revealed that heparin and citrate are the most commonly used anticoagulants during CRRT.Compared with heparin, RCA can effectively prolong the CRRT filter lifespan and reduce the risk of bleeding 2 .Recent randomized  www.nature.com/scientificreports/controlled studies have recommended the preferential selection of RCA over SHA in patients undergoing CRRT since it allows for a relatively longer filter lifespan 5,19 .However, the pre-thrombotic state was not considered in these studies.Accordingly, suitable anticoagulation strategies for managing patients with potential hypercoagulability due to elevated D-dimer levels prior to CRRT remain unclear.Since 2017, RCA has been preferentially selected at our unit for most patients requiring CRRT who lack contraindications to citrate.This has led to improvements in the CRRT anticoagulation effects and filter lifespan.However, in our clinical practice, we have also observed that the expected filter lifespan was not reached in certain pediatric patients with elevated D-dimer levels who underwent RCA-CRRT.D-dimer levels are associated with potential thrombosis and can effectively predict thrombotic complications 20,21 .Elevated D-dimer levels may be associated with an increased risk of thrombosis 22 , an increased tendency for CRRT filter clotting, and a shortened filter lifespan.These D-dimer levels have been identified as a risk factor for CRRT filter clotting 10 ; however, such a condition may be improved with heparin administration 23 .Based on the principles of anticoagulation, the concurrent use of heparin and citrate anticoagulation to exert effects on different pathways of the coagulation cascade may serve as a good anticoagulation strategy during CRRT for managing patients with elevated D-dimer levels.Ionized calcium acts as Factor IV in the coagulation cascade, and citrate can inhibit the coagulation process through reversible chelation of ionized calcium in the blood.Heparin can bind to antithrombin III (AT III) by inducing conformational changes in Factor Xa, considerably enhancing AT III activity, which leads to the inhibition of Factor Xa and Factor IIa activation in the coagulation cascade and ultimately produces an anticoagulant effect 24 .Therefore, the synergistic effect of citrate and heparin may be utilized to prolong the CRRT filter lifespan.However, there have been limited studies on the adoption of a combination citrate and heparin anticoagulation.Currently available research has focused on patients with COVID-19 who experienced premature extracorporeal clotting during CRRT due to a hypercoagulable state and involved the addition of heparin to citrate for the prolongation of the filter lifespan 10,11,25,26 .Valle et al. 10 reported that the CRRT filter lifespan in patients with COVID-19 was associated with D-dimer levels, with the risk of filter clotting increasing 0.94 times when D-dimer levels were ≥ 5990 ng/mL.The median filter lifespan was 25.6 h in patients with COVID-19 who underwent citrate anticoagulation only; however, it considerably increased to 81.9 h in patients with COVID-19 who underwent combination anticoagulation therapy with citrate and heparin.Additionally, the risk of filter clotting was decreased by 0.37 times with the addition of heparin.These previous findings are consistent with our findings.Specifically, the CRRT filter lifespan was 42.0 (37, 45.3) h, 38.0 (32.0, 41.0) h, and 46.0 (44.0, 49.0) h in the D− RCA only, D+ RCA only, and D+ RCA+ SHA groups, respectively.Therefore, systemic anticoagulation involving the addition of low-dose heparin to RCA may reduce the coagulation risk and prolong the CRRT filter lifespan in patients with elevated D-dimer levels.
Our findings indicated that the addition of low-dose heparin for systemic anticoagulation did not increase the incidence of bleeding episodes or demand for RBC transfusion in patients undergoing CRRT, which is consistent with the findings reported by Valle et al. 10 .This may be attributed to the relatively low heparin dose adopted in our study.
The CRRT filter lifespan is affected by multiple factors.For example, high ionized calcium levels are associated with a decreased filter lifespan 27 .In our study, the risk of filter clotting was increased when the post-filter ionized calcium level exceeded 0.4 mmol/L.Moreover, hemoglobin levels were negatively correlated with the filter lifespan, which is consistent with the findings in adult studies 10 .Approximately 33-65% of patients undergoing CRRT require blood transfusions [28][29][30] .Our results indicated that the CRRT filter lifespan was not associated with increased RBC transfusion, which is consistent with the findings reported by a case-control study 28 .A prospective study including pediatric patients undergoing RCA-CRRT demonstrated that both direct blood transfusion during CRRT and partial replacement of citrate transfusion (i.e., adoption of a reduced citrate infusion dose during blood transfusion in RCA-CRRT) were not associated with filter clotting during RCA-CRRT 31 .This could be attributed to the fact that the citrate content in blood products increases the total citrate dose for CRRT anticoagulation.This further reduces ionized calcium levels in the extracorporeal circuit, and thereby maintains effective anticoagulation without increasing the risk of filter clotting.Multivariate Cox regression analysis further revealed that the platelet count did not affect the CRRT filter lifespan.There remains no consensus on the effect of the platelet count on the filter lifespan.Certain studies have reported a negative correlation of the CRRT filter lifespan with a lower platelet count 32 , with the risk of filter clotting increasing when the www.nature.com/scientificreports/platelet count exceeds 45 × 10 9 /L 33 .However, another study has suggested a lack of association between platelet count and filter lifespan 34 .
To date, this is the largest study comparing between the anticoagulation strategies of RCA alone and RCA combined with SHA in patients other than those with COVID-19.Additionally, it is the first study of its kind in a pediatric population.
Our study has limitations.First, this was a single-center retrospective observational study with a limited sample size.CRRT initiation was determined solely at the discretion of attending physicians in the PICU, which may limit the universal applicability of our results.Second, D-dimer levels were used to assess the presence or absence of a hypercoagulable state.Furthermore, D-dimer levels are influenced by various factors and may not comprehensively and accurately reflect the pre-thrombotic state.Therefore, caution should be exercised when interpreting the study results.Finally, patients who underwent combination anticoagulation therapy in this study were not stratified based on D-dimer levels.Therefore, the optimal D-dimer level for the adoption of RCA combined with SHA remains unclear, and our results may not accurately reflect the relationship between D-dimer levels and the CRRT filter lifespan.

Conclusions
RCA combined with low-dose heparin anticoagulation during CRRT could reduce the risk of filter clotting and prolong the filter lifespan without increasing the risk of bleeding in patients at a high risk of hypercoagulability due to elevated D-dimer levels.

Figure 1 .
Figure 1.Flowchart of the patient selection process.

Figure 2 .
Figure 2. Filter lifespan.(a) Filter lifespan in the D− RCA only, D+ RCA only, and D+ RCA+ SHA groups shown as a boxplot with median and interquartile range.(b) Survival curves displaying filter lifespan among the D− RCA only, D+ RCA only, and D+ RCA+ SHA groups.
The most common indications for CRRT in descending order were sepsis, acute kidney injury, and cytokine storm.Table1presents the baseline characteristics of the patients.

Table 3 .
Cox regression analysis of factors affecting filter lifespan.CRRT continuous renal replacement therapy, RBC red blood cell.